The reverse transcriptase enzyme of HIV-1 remains one of the most important targets for therapeutic treatment of AIDS. One of the most difficult obstacles for successful treatment is the development of resistance by the enzyme to both nucleoside and nonnucleoside inhibitors. In order to gain insights into the molecular mechanisms of drug resistance, the three-dimensional structures of drug-resistant mutants of HIV-1 reverse transcriptase will be determined by the techniques of X-ray crystallography. Each of the mutants described has been cloned, and several of the mutants have already been crystallized. The laboratories of Dr. Edward Arnold (CABM/Rutgers) and Dr. Stephen Hughes (NCI-Frederick) have solved the structure of a ternary complex of HIV-1 RT, a double-stranded DNA template-primer, and a monoclonal antibody Fab fragment at 3.0 A resolution, and the structure is being refined at 2.8 A resolution. Most of the mutant structures will be determined using this crystal form and convenient difference Fourier techniques. Where possible, the structures of nucleoside-resistant mutations will also be studied with bound triphosphate forms of nucleoside inhibitors. Some of the studies with nonnucleoside-resistant mutations will utilize a crystal form containing HIV-1 RT with bound nonnucleoside inhibitors that diffracts X-rays to a resolution of 2.6 A. Specifically, the structures of mutants resistant to AZT (with changes at residues 41, 67, 70, 215, 219), ddI (74 and 184), ddGTP (89), and a variety of nonnucleoside inhibitors (100, 103, 106, 181, 188, 236) will be determined. We will study mutations that, when added to an AZT- resistant genotype, lead to a return of AZT sensitivity (such as 74 added to the five AZT-resistant mutations, and 181 added to 215). We are prepared to study additional combinations that become identified as being clinically significant. Finally, we propose to determine the structure of a complex of a TIBO nonnucleoside inhibitor with a mutant (P236L) that is BHAP-resistant but hypersensitive to TIBO and other nonnucleoside RT inhibitors. These studies should provide a three-dimensional basis for understanding the molecular mechanisms of HIV-1 RT drug resistance. The known structure of HIV-1 RT in complex with template-primer has permitted placement of drug-resistant mutations in a three-dimensional context, but in order to fully interpret RT drug resistance, it will be valuable to know the precise changes caused by the amino acid changes (loss of contact, steric conflict, conformational changes of protein, nucleic acids, or both, etc). It is hoped that this information may contribute to the development of inhibitors or strategies that can slow or even overcome the development of clinical resistance to antiviral treatments of AIDS.